1 Induction of Endoplasmic Reticulum Stress by Sorafenib and Activation Of

Author Manuscript Published OnlineFirst on December 13, 2012; DOI: 10.1158/1535-7163.MCT-12-0532 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Induction of endoplasmic reticulum stress by sorafenib and activation of NF-κB

by lestaurtinib as a novel resistance mechanism in Hodgkin lymphoma cell

lines

Meike Stefanie Holz1, Angela Janning1, Christoph Renné2, Stefan Gattenlöhner3, Tilmann

Spieker1, Andreas Bräuninger1,3

1 Gerhard-Domagk-Institute for Pathology, Westfälische Wilhelms-University, Münster,

Germany

2 Department of Pathology, Johann Wolfgang Goethe-University, Frankfurt, Germany

3 Department of Pathology, Justus-Liebig-University, Giessen, Germany

Running title: Effects of sorafenib and lestaurtinib on Hodgkin cell lines

Key words: Hodgkin lymphoma, receptor tyrosine kinase, tyrosine kinase inhibitor,

signaling pathways, NF-κB activation

Correspondence: Andreas Bräuninger, PhD

Department of Pathology, Justus-Liebig-University Giessen

Langhansstr. 10, 35392 Giessen, Germany

Phone: ++49 641 98541130; Fax: ++49 641 98541139

E-mail: [email protected]

The Authors disclose no potential conflicts of interest.

Word count: 4996

Total number of tables and figures: 6

Downloaded from mct.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 13, 2012; DOI: 10.1158/1535-7163.MCT-12-0532 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoman (HL) show aberrant

expression and activation of several receptor tyrosine kinases (RTKs) in the majority of

cases. Therefore we tested whether tyrosine kinase inhibitors (TKIs) already in clinical use or

late stages of clinical trials have antiproliferative effects on HRS cell lines and evaluated the

targets, affected signaling pathways and mechanisms of cell death and resistance. Sorafenib

and lestaurtinib had antiproliferative effects on HRS cell lines at concentrations achievable in

patients. Sorafenib inhibited PDGFRα, TRKA and RON, caused decreases in total and

phosphorylated amounts of several signaling molecules and provoked Caspase 3

independent cell death, most likely due to endoplasmic reticulum stress as indicated by

upregulation of GADD34 and GADD153 and phosphorylation of PERK. Lestaurtinib inhibited

TRKA, PDGFRα, RON and JAK2 and had only a cytostatic effect. Besides deactivation

lestaurtinib caused also activation of signaling pathways. It caused increases in CD30L and

TRAIL expression, and CD30L/CD30 signaling likely led to the observed concomitant

activation of ERK1/2 and the alternative NF-κB pathway. These data disclose the possible

utility of sorafenib for the treatment of HL and highlight NF-κB activation as a potential novel

mechanism of resistance towards tyrosine kinase inhibitors.

Introduction

In HRS cells of HL the activation of several signaling pathways is not only caused by

mutations but also by the activation of receptors like receptor tyrosine kinases (RTK) by

ligands expressed by cells of the microenvironment or the HRS cells themselves (1-4). Thus,

HRS cells express eight RTKs, PDGFRα, DDR2, EPHB1, RON, TRKA, TRKB, TIE1 and

CSF1R, in varying patterns and PDGFRα and TRKA have been shown to be activated in

primary cases (5-7). As mutations affecting RTKs could not be detected in HRS cell lines and

as activating ligands were frequently coexpressed in HRS cells or expressed by surrounding

cells in the infiltrate of primary HL cases (5), the receptors are presumed to be activated in

an autocrine or paracrine fashion.

Several inhibitors of tyrosine kinases (TK) are highly effective in the treatment of

malignancies with aberrantly activated TKs (8). Tyrosine kinase inhibitors (TKIs) are usually

most effective in malignancies with mutation activated TKs such as lung cancer with mutated

EGFR treated with gefitinib (9). However, several TKIs are also used for the treatment of

cancers in which so far no TK activating mutations have been identified like sorafenib in

advanced renal cell carcinoma and hepatocellular carcinoma (10-12).

In previous experiments the PDGFRα inhibitor imatinib and K-252a (5, 13), an

inhibitor of TRKA, caused decreases of HRS cell proliferation indicating that also HRS cells

could be sensitive to TK inhibition although so far no TK activating mutations have been

observed. Therefore we chose nine TKIs, either already approved for cancer therapy or in

late stages of clinical trials, to examine their effects on HRS cell lines. We show here that

sorafenib and lestaurtinib reduced HRS cell proliferation significantly at concentrations

achievable in patients, in line with recently published studies (14, 15). As sorafenib induced

cell death without activation of the effector Caspase 3 and as lestaurtinib, in contrast to a

recently published study (15), affected cell growth but did not induce cell death, we analyzed

the targets, the impact on activated signaling pathways and the modes of cell death induction

and resistance of both TKIs.

Materials and methods

Cell lines

HRS cell lines (HDLM-2, KM-H2, L-428, L-1236 and U-H01) were obtained from the DSMZ

(Braunschweig, Germany) and cultured as recommended. Cell line identity was determined

by the DSMZ by STR DNA fingerprinting and ensured by continuous culture for less than six

months. Haematopoietic neoplasm U-2932, SU-DHL-8, SU-DHL-10, KARPAS-422, SU-DHL-

1, KARPAS-299, DG-75, RAMOS, DAUDI, RAJI, JURKAT, L-363, K-562 and HL-60,

carcinoma MCF7, MDA-MB-468, A-549, HTB-56, HELA, and fibroblast NIH-3T3 cell lines

were obtained from several colleagues (R. Küppers, Essen, Germany; C. Müller-Tidow,

Münster, Germany; A. Neumann, Münster, Germany). As they were used only for

comparisons of the antiproliferative effects of sorafenib and lestaurtinib they were not

authenticated by the authors. Numbers of viable cells were determined with a CASY TT cell

counter as technical duplicates (Innovatis AG, Reutlingen, Germany).

MTT assay for metabolic activity

Cells were plated at 1x105 cells/ml (L-428 and KM-H2) or 2x105 cells/ml (L-1236) and

incubated with the indicated drug or solvent alone. To quantify enzymatic reductase activity

corresponding to metabolic activity, the “In Vitro Toxicology Assay” (Sigma-Aldrich) was used

and the concentration of the dye converted from MTT was measured in duplicate at 570 nm

with a NanoDrop (Peqlab, Erlangen, Germany).

Apoptosis analysis by flow cytometry

For analysis of cell death induction after treatment with lestaurtinib or sorafenib, L-428, L-

1236 and KM-H2 cells were stained with FITC-AnnexinV (BD Biosciences Pharmingen, San

Jose, CA) and propidiumiodide (PI) (AppliChem, Darmstadt, Germany) according to the

manufacturer's instructions and analyzed by flow cytometry.

Immunoprecipitation of RTKs

Cells were lysed in NP-40-based lysis buffer (Pierce Co-Immunoprecipitaion Kit; Thermo

Scientific, Rockford, IL) supplemented with 2 mM Na3VO4 and cOmplete Mini Protease

Inhibitor Cocktail (Roche Applied Science). Cell lysates (250 µg protein) were incubated with

1 µg of anti-Ron β (C-20) or anti-Trk (C-14) antibodies (Santa Cruz Biotechnology, Santa

Cruz, CA) and immune complexes were collected by binding to Dynabeads® Sheep anti-

Rabbit IgG (Life Technologies, Carlsbad, CA). Proteins were eluted by boiling Dynabeads in

SDS sample buffer.

Western blot analysis

Western blot analysis was performed as described in Supplementary Materials and methods

with primary antibodies specified in Supplementary Table S1. Results shown here are

representative for at least two independent experiments. Densitometric analysis was

performed with the open access image processing program ImageJ.

Reverse transcription-PCR and quantitative real time PCR (qRT-PCR)

Total RNA was extracted with the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany).

Complementary DNA was synthesized using the 1st Strand cDNA Synthesis Kit (Roche

Applied Science). For quantitative PCR analysis, the Applied Biosystems® Power SYBR®

Green Master Mix System (Life Technologies) was utilized. Technical duplicates were

performed for each sample. Primer sequences are given in Supplementary Table S2. Identity

of all PCR products was verified by sequencing.

Affymetrix GeneChip Human 1.0 ST Array analysis

KM-H2 was incubated for 24 h with 1 µM lestaurtinib or DMSO and total RNA was extracted

and analyzed with GeneChip Human 1.0 ST arrays. Processing of the microarrays and initial

bioinformatic analyses were performed by the Integrated Functional Genomics (IFG) Unit of

the Interdisciplinary Center for Clinical Research (IZKF) at the Medical Faculty of the

University of Münster, Germany. After scaling data were imported into GeneSpringGX

(Agilent Technologies, Santa Clara, CA) and normalized using the default normalization

methods. Mean values of replicate gene arrays were used to determine changes in mRNA

expression by pairwise comparison (p < 0.05) of lestaurtinib treated samples to control

samples. To identify gene ontology (GO) categories, differentially expressed genes (Fold

change > 2) were analyzed with GeneCodis (16). All data regarding the microarray analysis

have been deposited at ArrayExpress under accession number E-MEXP-3565.

Results

Sorafenib and lestaurtinib inhibit proliferation of HRS cell lines

Screening of nine TKIs revealed that only sorafenib and lestaurtinib reduced proliferation of

three of five HRS cell lines after 48 h in a concentration dependent manner and at

concentrations achievable in patients (IC50 for sorafenib: 8 µM for L-1236 and L-428; 5 µM

for KM-H2; IC50 for lestaurtinib: 10 nM for L-1236; 300 nM for L-428 and KM-H2) (Fig. 1A-E

and Supplementary Tables S3 and S4) (17, 18). At longer incubation times also HRS cell

lines HDLM-2 and U-HO1 showed sensitivities comparable to other HRS cell lines (Fig. F-G),

most likely due to their longer doubling times. These sensitivities were in line with two

recently published studies for HRS cell lines (14, 15). Incubation of other lymphoma,

leukemia and carcinoma cell lines with 5 µM sorafenib, a concentration at which other cell

lines without RTK mutations showed sensitivity (19-21), and 300 nM lestaurtinib, a

concentration at which cell lines with FLT3 or JAK2 mutations were sensitive (22, 23),

revealed that sensitivities towards the multi-kinase inhibitor sorafenib were comparable for all

cell lines (Fig. 1F), and that HRS cell lines were less sensitive towards lestaurtinib than all

other tested cell lines (Fig. 1G).

Cell death induction by sorafenib but not lestaurtinib in HRS cell lines

As sorafenib and lestaurtinib inhibited growth of HRS cell lines, we determined whether this

was due to a cytostatic effect or cell death. Therefore we incubated L-428, L-1236 and KM-

H2 cells with the TKIs and utilized Western blot (WB) for active, cleaved forms of the effector

Caspase 3 and flow cytometry analysis after annexinV/PI staining to detect apoptosis (Fig.

2).

Sorafenib treatment lead to a substantial decrease of viability in three HRS cell lines

as detected by annexinV/PI staining, but not by WB for activation of the effector Caspase 3 in

L-428 and KM-H2. Lestaurtinib induced apoptosis in L-1236 as detected by annexinV/PI

staining and Caspase 3 WB but had, in contradiction to previously reported findings (15),

only a cytostatic effect on L-428 and KM-H2 cells as no substantial apoptosis induction could

be detected by either means.

Targets of sorafenib and lestaurtinib in HRS cell lines

To identify the targets of sorafenib and lestaurtinib in HRS cell lines, we analyzed their

effects on RTK phosphorylation via direct WB or WB after immunoprecipitation (IP) with

antibodies against p-TRK, p-PDGFRα and p-RON. The relative phosphorylation of RTKs was

calculated in relation to the total amount of RTK detected by densitometry analysis of

representative WBs (Supplementary Table S5).

The effects of TKIs on TRKA (hereafter referred to as TRK) were analyzed with L-

1236 and HDLM-2, which showed that when pre-treated with sorafenib or lestaurtinib, β-NGF

failed to induce TRK phosphorylation in both cell lines (Fig. 3A, Supplementary Table S5).

In L-428 RON is significantly phosphorylated without application of its ligand, most

likely due to the presence of a constitutively active splice variant lacking the fourth

extracellular immunoglobulin-plexin-transcription domain (data not shown) (24). Sorafenib

nearly completely abolished the phosphorylation of RON in L-428 (Fig. 3B), and also

lestaurtinib had an inhibitory effect on the phosphorylation of RON (Fig. 3B and

Supplementary Table S5).

In U-HO1, ligand induced phosphorylation of PDGFRα was decreased by sorafenib and also

by lestaurtinib (Fig. 3C, Supplementary Table S5). Surprisingly, expression and

phosphorylation of PDGFRα was increased in KM-H2 cells when treated with lestaurtinib

(Fig. 3D), while evaluation of the effects of sorafenib in KM-H2 was difficult due to the low

PDGFRα phosphorylation under normal culture conditions. After pre-incubation with

lestaurtinib, sorafenib caused a decrease of PDGFRα phosphorylation and protein amount

(Fig. 3E).

Taken together, the known targets of sorafenib and lestaurtinib, PDGFRα and TRK,

respectively, could be verified as targets in HRS cell lines while TRK and RON were

identified as new targets of sorafenib and PDGFRα and RON as new targets of lestaurtinib.

Sorafenib and lestaurtinib reduce activation of multiple intracellular signaling pathways

To analyze the effect of TKIs on signaling pathways usually activated by RTKs, WB analysis

was performed. Treatment with sorafenib caused the reduction of phosphorylation of JAK2,

various STATs and RAF1 in all three cell lines and reduction of AKT1 in L-1236 and KM-H2.

However, in all instances the reduced phosphorylation was accompanied by concomitant

reduction of total protein amounts (Fig. 4A). ERK1/2 were less phosphorylated in all cell lines

without decreases in total protein amounts.

Lestaurtinib caused decreases in phosphorylation of AKT1, JAK2 and RAF1 in all cell

lines with concomitant reduction of RAF1 total protein amount (Fig. 4B). The phosphorylation

levels of STATs were reduced in L-428 and L-1236. Unexpectedly, the phosphorylation of

ERK1/2 was increased in all three cell lines and the phosphorylation of STATs was constant

in KM-H2 after lestaurtinib treatment with elevated total protein amounts of JAK2 and STATs

in this cell line.

Sorafenib provokes endoplasmic reticulum stress in HRS cell lines

Sorafenib can cause endoplasmic reticulum (ER) stress with a general reduction of

translation and Caspase 3 independent cell death in human leukemia cells (25), and ER

stress could thus also account for the reduction in total protein amounts of several signaling

molecules and Caspase 3 independent cell death in HRS cell lines. Therefore we analyzed

the effect of sorafenib on different ER stress markers in HRS cell lines by WB and qRT-PCR.

The phosphorylation of PERK, one of the ER resident proteins inducing the stress response

(26), and the expression of genes induced by ER stress, GADD34 and GADD153 (25-27),

were monitored (Fig. 5).

All three analyzed cell lines had phosphorylated PERK when incubated with solvent

alone, which increased upon incubation with 10 µM sorafenib in L-428 and KM-H2.

Incubation with 10 µM sorafenib increased GADD34 and GADD135 mRNA levels in all cell

lines as determined by q-RT-PCR (7.5 to 16 fold and 14 to 37 fold increases for GADD34

and GADD135, respectively). To investigate whether the induction of ER stress by sorafenib

was a HRS cell specific effect, the expression of GADD34 and GADD153 was also analyzed

in HDLM-2, U-HO1 and two non-Hodgkin lymphoma cell lines with comparable sensitivity

towards the TKI, L-363 and RAMOS. In all cell lines 24 h incubation with 10 µM sorafenib

caused an increase of GADD34 and GADD135 mRNA levels comparable to the effect in

HRS cell lines (Supplementary Fig. S1).

These observations indicate that sorafenib provokes ER stress, which may cause cell

death, in HRS and the other cell lines tested. The induction of ER stress could thus be a

general effect of sorafenib.

Lestaurtinib activates the alternative NF-κB pathway in HRS cell lines

Despite the dephosphorylation of the anti-apoptotic AKT1 and in contrast to a recent study

(15), lestaurtinib did not induce apoptosis in L-428 and KM-H2 in our hands. The lestaurtinib

induced ERK1/2 phosphorylation in all HRS cell lines and increased expression of PDGFRα,

JAK2 and STATs in KM-H2 indicated that the expression and activation of several signaling

pathway constituents, which could support survival, increased upon AKT1 and JAK inhibition

in HRS cell lines. To address this issue systematically, we performed differential genome

wide gene expression analysis after lestaurtinib treatment of KM-H2.

KM-H2 cells were incubated with 1 µM lestaurtinib or solvent for 24 h and differentially

expressed transcripts were identified using replicates of Affymetrix GeneChip Human 1.0

Arrays and the GeneSpringGX software. Sixty-three genes were at least twofold up- and 180

genes were at least twofold down-regulated (Supplementary Tables S6 and S7). Gene

ontology analysis revealed the affiliation of most (116 of 180) down-regulated genes to

biological processes linked to cell division and proliferation (Supplementary Table S8) and

most of these genes enhance cell cycle progression. The upregulated genes showed no

strong affiliation to specific biological processes.

We chose seven upregulated genes which could enhance cell survival (LIPH, PIM2,

SOS2, STAT4, TNFSF8, TNFSF10 and TRAF3IP3) for further analysis by qRT-PCR with

KM-H2, L-428 and L-1236 (Fig. 6A). For KM-H2, increases in RNA levels of all genes could

be confirmed, demonstrating the validity of the microarray analysis. The most distinctly

induced genes in all three cell lines were TNFSF8 (6 to 18 fold), the CD30 ligand, and

TNFSF10 (3 to 13 fold), the TRAIL-R ligand.

CD30, a cell surface tumor necrosis factor receptor (TNFR) expressed by most HRS

cells and HRS cell lines (28), has been shown to activate the MEK/ERK pathway and the

classical and alternative NF-κB pathways upon binding of its ligand (29-31). The observed

increases in ERK1/2 phosphorylation upon lestaurtinib treatment are thus most likely due to

the CD30L upregulation. To determine whether the lestaurtinib induced upregulation of

CD30L resulted also in NF-κB activation we analyzed the expression of transcriptional

targets of the classical NF-κB pathway (ICAM1, LTA and BIRC3) by qRT-PCR, but observed

no increases in expression (data not shown). In addition, we analyzed activation of the

alternative NF-κB pathway by monitoring the accumulation of p52 by WB. After 24 h

incubation with 1 µM lestaurtinib increased amounts of p52 were detected in all three cell

lines (Fig. 6B).

To investigate whether the lestaurtinib induced upregulation of CD30L was a more

general mechanism, two further HRS and anaplastic large cell lymphoma (ALCL) cell lines

expressing also CD30 were analyzed by q-RT-PCR for CD30L mRNA changes after 24 h

treatment with 1 µM lestaurtinib (Supplementary Fig. S2). An increase of CD30L mRNA

amounts could be observed in one of the HRS cell lines but not in the second HRS cell line

nor in the ALCL cell lines, indicating that the upregulation of CD30L is a HRS cell specific

resistance mechanism.

Taken together, lestaurtinib induced upregulation of CD30L and TRAIL in HRS cell

lines and the former most likely caused ERK1/2 phosphorylation and activation of the

alternative NF-κB pathway.

TKIs can increase the effect of conventional chemotherapeutic drugs

The combination of conventional chemotherapeutic drugs with TKIs is a strategy to enhance

the effect of cancer therapy (32). For example the EGFR inhibitor erlotinib is added to

conventional chemotherapy for patients with advanced-stage pancreatic cancer (33).

Therefore we examined the effect of sorafenib and lestaurtinib on HRS cell lines in

combination with drugs used in conventional HL chemotherapy.

The metabolic activity of L-428, L-1236 and KM-H2 cells was measured (MTT assay)

after 72 h treatment with vinblastine and doxorubicin alone, sorafenib or lestaurtinib alone, or

the combination of one chemotherapeutic with one TKI (Supplementary Fig. S3). The

inhibitory effects of vinblastine and doxorubicin were in general enhanced by the

simultaneous treatment with sorafenib or lestaurtinib compared to either TKI or vinblastine or

doxorubicin alone. This effect was most evident for the combination of sorafenib with either

conventional chemotherapeutic drug in L-428 and KM-H2 cells.

Discussion

Several different RTKs are expressed and activated in HRS cells of HL (5), and the inhibition

of these RTKs with TKIs could be a novel option for treatment. In a screening of nine TKIs

we found that all HRS cell lines tested were sensitive towards the TKIs sorafenib and

lestaurtinib at concentrations achievable in patients.

Sorafenib, a multi-kinase inhibitor induced cell death of L-428, L-1236 and KM-H2

after prolonged incubation. Though other studies investigating sorafenib observed

antiproliferative activity at low nanomolar concentrations when tumor cell lines with mutation

activated RTKs were examined (34), the sensitivities observed for HRS cell lines were

comparable to what we found for other cell lines and what was found in other studies,

analyzing for example carcinoma or other lymphoma cell lines (19-21), and is in line with a

recently published study analyzing the effect of the TKI on HRS cell lines (14). Sorafenib

inhibited not only the activation of its known targets RAF1 and PDGFRα (35, 36), but also

TRK and RON and thus at least two of the kinases tested here in each HRS cell line, and,

given its broad effects on several different cell lines, likely also other kinases not tested here.

The sorafenib induced deactivation of intracellular signaling molecules was often

accompanied by losses of total amounts of these proteins, and sorafenib can cause ER

stress with reduced translation in leukemia cells (25). As we observed typical features of ER

stress also in the sorafenib treated HRS cell lines it is likely that the losses of total amounts

of signaling molecules are due to ER stress induced reduction in translation combined with

their fast turnover rates. Furthermore, ER stress caused cell death in a Caspase 3

independent way in leukemia cells (25), and also the sorafenib induced cell death in L-428

and KM-H2 was independent of Caspase 3 activation. We conclude that cell death in L-428

and KM-H2 might be induced by the same ER stress dependent mechanisms as in leukemia

cells (25). As induction of ER stress markers in response to sorafenib treatment was also

observed in two other non-Hodgkin lymphoma cell lines, induction of ER stress by sorafenib

may be a more general phenomenon.

The sensitivity of HRS cells towards lestaurtinib is comparable to what was reported

for acute myeloid leukemia cells dependent on mutant FLT3 activity (22), or

myeloproliferative neoplasm cell lines with the constitutively active JAK2 mutant V617F (23).

However, also all other lymphoma, leukemia and carcinoma cell lines tested here were even

more sensitive than the HRS cell lines, which could be either due to the interaction of

lestaurtinib with a broad kinase target spectrum, as recently supposed (37), or dependency

of all tested cell lines on JAK/STAT signaling. Lestaurtinib was recently reported to induce

Caspase 3 activity in some HRS cell lines (15). Though exhibiting strong cytostatic effects,

we observed an induction of apoptosis by lestaurtinib only in the already pre-apoptotic L-

1236, in contrast to the Caspase 3 activation in L-428 observed by Diaz et al. (15).

Lestaurtinib inhibited activation of its known targets TRK and JAK2 and

phosphorylation of AKT1 in all and phosphorylation of several STATs in L-428 and L-1236.

Furthermore it inhibited ligand induced phosphorylation of PDGFRα in one cell line and

reduced the constitutive phosphorylation of RON in L-428. In KM-H2, however, lestaurtinib

treatment caused increases in total amounts of JAK2 and STATs and the amounts of

phosphorylated STAT3 and 5 remained constant despite complete JAK2 dephosphorylation.

We observed also increases in expression and phosphorylation of PDGFRα in KM-H2 upon

lestaurtinib treatment and thus it is conceivable that the activated PDGFRα directly or via

activation of other intracellular TKs, e.g. SRC family kinases, contributed to the STAT

phosphorylation under lestaurtinib treatment (38). Such maintenance of activation of a

signaling pathway on which tumor cells are “dependent” by increased expression and

activation of another TK upon pharmacological inhibition of the “driver” TK is a known

mechanism of secondary TKI resistance.

Another way of tumor cells to escape the pharmacological inhibition of antiapoptotic

signaling pathways is the activation of alternative pathways (39-41). As lestaurtinib treatment

caused deactivation of AKT1, whose activity is usually essential for HRS cell survival (42,

43), without inducing apoptosis, we performed a systematic screen for upregulated

constituents of other signaling pathways and found that lestaurtinib caused significant

increases of CD30L and TRAIL expression in all three analyzed HRS cell lines.

The activation of CD30 by CD30L has previously been shown to cause ERK

phosphorylation and NF-κB activation in HRS cell lines (29-31), and, indeed, we observed

increased ERK1/2 phosphorylation and increases in the activity of the alternative NF-κB

pathway upon lestaurtinib treatment in all analyzed HRS cell lines. TRAIL usually induces

apoptosis in tumor cells via the TRAIL-R associated DISC. However, in HRS cells apoptosis

induction via TRAIL-R associated Caspase 8 is prevented by the strong c-FLIP expression

(44, 45), and in cancer cells resistant towards TRAIL induced apoptosis, TRAIL increased

survival in a NF-κB dependent fashion (46). It is thus conceivable that in HRS cell lines the

lestaurtinib induced increases in TRAIL expression also contribute to NF-κB activation.

Furthermore, activation of the alternative NF-κB pathway may also be increased by the

observed TRAF3IP3 upregulation, which by releasing NIK from TRAF3 may cause activation

of IKKα with subsequent p100 processing (47, 48).

It is thus likely that in HRS cells the inhibitory effects of lestaurtinib on the survival

enhancing AKT1 are compensated by activation of CD30 signaling via CD30L upregulation

and activation of ERK1/2 and NF-κB and also by TRAIL mediated NF-κB activation. As the

combined application of the MEK/ERK inhibitor PD0325901 with lestaurtinib did not result in

apoptosis (data not shown), activation of NF-κB pathway may be the more relevant

mechanism of apoptosis prevention. Interestingly, this effect seems to be HRS cell specific,

as no lestaurtinib induced upregulation of CD30L transcription was observed in other CD30

expressing cell lines.

The addition of TKIs to cancer treatment with conventional chemotherapeutic drugs

has beneficial effects in several instances. Therefore we combined lestaurtinib and sorafenib

with doxorubicin and vinblastine and indeed observed increased inhibitory effects on

metabolic activity compared to the single agents.

The combination of conventional chemotherapeutic drugs with sorafenib showed

additive inhibitory effects in those cell lines possibly undergoing completely Caspase 3

independent cell death in response to sorafenib, L-428 and KM-H2. Apoptosis via Caspase 3

is one major cell death pathway caused by conventional chemotherapeutic agents (49).

Potentially the combination of the two drugs is most efficient when different mechanisms of

cell death are triggered, the apoptotic pathway through Caspase 3 by a conventional

chemotherapeutic agent and Caspase independent cell death by sorafenib.

Combining lestaurtinib with conventional chemotherapeutic drugs did not significantly

alter the effect of lestaurtinib alone in L-428 and KM-H2. Here, lestaurtinib mediated NF-κB

activation may provide protection from moderate concentrations of conventional

chemotherapeutics.

In summary, we show here that lestaurtinib inhibits proliferation and sorafenib induces

cell death in several HRS cell lines. The sorafenib induced cell death was Caspase 3

independent, in line with the induction of ER stress which can cause cell death via Caspase

3 independent mechanisms. The combination of sorafenib with conventional

chemotherapeutics had additive effects, likely due to the induction of cell death by two

different mechanisms. This indicates a potential use of sorafenib in HL therapy in

combination with conventional chemotherapeutic drugs. Regarding the effects of lestaurtinib,

our results indicate that HRS cells can escape apoptosis by TKI mediated AKT1 inhibition via

increases in the TNFR mediated activation of the alternative NF-κB pathway. This mode of

TKI resistance differs from the usually observed upregulation of other TKs or increased

signaling via other TK activated pathways and seems to be a HRS cell specific resistance

mechanism.

Acknowledgements

We would like to thank Inka Buchroth and Sandra Mühleis for excellent technical assistance.

Grant support

This work was supported by the Deutsche Forschungsgemeinschaft (grant no. BR1238/6-3

A. Bräuninger).

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Figure legends

Figure 1. Sensitivity of Hodgkin-Reed/Sternberg cell lines to tyrosine kinase inhibitors. A,

Chemical structures of compounds used in this study. B, HRS cell lines L-1236 and L-428

(both expressing TRKA, RON, DDR2 and EPHB1), KM-H2 (expressing PDGFRα and

DDR2), HDLM-2 (expressing TRKA, DDR2 and EPHB1) and U-HO1 (expressing PDGFRα

but not TRKA and RON) were incubated with different TKIs. Percent of viable cells after 48 h

treatment with 5 µM of TKI are shown as mean values of two experiments. Reductions below

80% were considered relevant. For lapatinib, sunitinib and dasatinib see Supplementary

Table 4. C-E, For generation of dose-response curves for sorafenib and lestaurtinib, cells

were incubated with the indicated concentrations of the respective TKI for 72 h and counted

(C: sorafenib; D: lestaurtinib). Additionally, the metabolic activity (E) of HRS cell lines treated

with a concentration range of lestaurtinib for 72 h was measured with a MTT assay. Mean

values and standard deviations were calculated from three independent experiments. F-G,

Different lymphoma, leukemia and carcinoma cell lines were incubated with (F) 5 µM

sorafenib or (G) 300 nM lestaurtinib for 72 h and counted. Shown are the mean values from

two experiments.

Figure 2. Assessment of cell death induction in Hodgkin-Reed/Sternberg cell lines by

sorafenib and lestaurtinib. A, Induction of cell death was monitored by WB for Caspase 3 and

its active cleaved form after 24 h and 72 h (longer film exposure) treatment with 1 µM

lestaurtinib or 10 µM sorafenib. B, Flow cytometry analysis of annexinV-FITC and propidium

iodide (PI) stained cells was performed after 24 h and 72 h incubation with DMSO, 1 µM

lestaurtinib or 10 µM sorafenib. C, Representative scatter plots of an annexinV-FITC/PI flow

cytometry analysis for L-428 treated with DMSO or sorafenib for 72 h. Note that equal

numbers of events were counted, most of which located close to the x-axis in the DMSO

control scatter plot example. The percentage of cells stained for annexinV and/or PI was

calculated from two independent experiments.

Figure 3. Western blot analysis of receptor tyrosine kinase phosphorylation after incubation

with sorafenib or lestaurtinib. A, WB analysis of immunoprecipitated TRK after 4 h incubation

of cells in 1% FCS to increase induction of RTK phosphorylation, 10 min pre-incubation with

TKIs sorafenib (10 µM) or lestaurtinib (1 µM) and stimulation with 25 ng/ml β-NGF for 2 min.

B, WB analysis of immunoprecipitated RON after 24 h treatment with 10 µM sorafenib or 1

µM lestaurtinib. C, WB analysis of PDGFRα after cells were serum starved and pre-treated

with sorafenib or lestaurtinib as described for Figure 3A and stimulated with 100 ng/ml

PDGFAA for 2 min. D, WB analysis of PDGFRα in KM-H2 after 24 h incubation with 1 µM

lestaurtinib or 10 µM sorafenib. E, WB analysis of PDGFRα in KM-H2 cells pre-treated with 1

µM lestaurtinib for 24 h and subsequent incubation with 10 µM sorafenib for 10 min or 1 h. All

experiments were performed at least twice.

Figure 4. Western blot analysis of intracellular signaling pathways after tyrosine kinase

inhibitor treatment. Shown are WBs for lysates after 24 h treatment with (A) 10 µM sorafenib

or (B) 1 µM lestaurtinib compared to untreated cells. AKT1, JAK2, STATs 3, 5 and 6, RAF1

and ERK1/2 as well as their active phosphorylated forms were detected.

Figure 5. Endoplasmic reticulum stress after sorafenib treatment. L-1236, L-428 and KM-H2

were treated with sorafenib. A, WB analysis of PERK phosphorylation after 24 h (L-1236 and

L-428) or 12 h (KM-H2) incubation with 10 µM sorafenib or the solvent DMSO. B, mRNA

amounts of GADD34 and GADD153 measured by qRT-PCR, normalized to the

housekeeping gene PPIA and shown as fold changes compared to control. L-1236, L-428

and KM-H2 cells were incubated with 10 µM sorafenib for 24 h. Mean values and standard

deviations were calculated from two independent experiments.

Figure 6. Changes of mRNA expression of selected genes in Hodgkin-Reed/Sternberg cell

lines and activation of the alternative NF-κB pathway after lestaurtinib treatment. A, L-1236,

L-428 and KM-H2 cells were incubated with 1 µM lestaurtinib for 24 h. mRNA amounts of

LIPH, PIM2, SOS2, STAT4, TNFSF8, TNFSF10 (Variant 1/2), TNFSF10 (Variant 3), and

TRAF3IP3 were measured by qRT-PCR, normalized to the housekeeping gene PPIA and

are shown as fold changes compared to control. Values of samples with a Ct > 35 or

undetectable amplification for some of the DMSO control samples of L-428 and L-1236 for

TNFSF8 and TNFSF10 were set to 35 for ΔΔCt calculation and the given fold changes may

thus be an underestimation. Mean values and standard deviations were calculated from two

independent experiments. B, WB analysis of p52 accumulation in HRS cell lines after

incubation with 1 µM lestaurtinib or DMSO for 24 h.

Induction of endoplasmic reticulum stress by sorafenib and activation of NF- κB by lestaurtinib as a novel resistance mechanism in Hodgkin lymphoma cell lines

Meike Stefanie Holz, Angela Janning, Christoph Renne, et al.

Mol Cancer Ther Published OnlineFirst December 13, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-12-0532

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